Within the higher eukaryotic chromosome, DNA is folded into multiple levels of organization. The highest levels of chromatin folding, consisting of the folding of 30 nm chromatin fibers, account for up to a 250:1 packing ratio and involve the organization of entire transcription and replication functional domains and their compaction within mitotic and interphase chromatids. It is this large-scale chromatin structure which is the focus of our research. Our long term objectives are to determine the basic folding motifs underlying large-scale chromatin structure and chromosome architecture, and the relationship of chromatin structure at this level of organization to regulation of DNA function, including transcription and replication. Our specific aims are to answer the following questions: l) Do early events in mitotic chromosome condensation (decondensation ) correspond to a folding (unfolding) of chromonema fibers; 2) What is the compaction ratio for chromonema fibers; what chromatin folding motifs underlie the structure of chromonema fibers? 3) What is the reproducibility of large- scale chromatin folding? 4) What changes in large-scale chromatin organization accompany DNA replication and transcription? 5) Is accessibility of trans factors limited by large-scale chromatin organization? A recently developed method for in situ localization of specific chromosome regions using lac operator - repressor binding provides excellent ultrastructural preservation and will allow us to visualize specific chromosome regions in living cells by light microscopy and at high resolution using electron microscopy. Analysis of folding motifs underlying large-scale chromatin organization will exploit three- dimensional reconstructions of interphase chromosome structure at overlapping resolution generated using optical sectioning light microscopy, electron microscopy tomography, serial sectioning electron microscopy, and a combination of serial semi-thick serial sections and electron microscopy tomography. This proposed research will provide a basic description of the folding motifs underlying large-scale chromatin and chromosome organization. Future directions of our work will focus on the structural and mechanistic links between transcription and large-scale chromatin organization, particularly at the level of chromatin domains, tens to hundreds of kb in length.